Arachidonic acid (ARA; cis-5, 8, 11, 14-eicosatetraenoic; ω-6) is an important precursor in the production of eicosanoids (e.g., prostaglandins, thromboxanes, prostacyclin and leukot). Additionally, ARA is recognized as: (1) an essential long-chain polyunsaturated fatty acid (PUFA); (2) the principal ω-6 fatty acid found in the human brain; and, (3) an important component of breast milk and many infant formulas, based on its role in early neurological and visual development. Although adults obtain ARA readily from the diet in foods such as meat, eggs and milk (and can also inefficiently synthesize ARA from dietary linolenic acid (LA)), commercial sources of ARA oil are typically produced from natural vegetarian sources (e.g., microorganisms in the genera Mortierella (filamentous fungus), Entomophthora, Pythium and Porphyridium (red alga)). Most notably, Martek Biosciences Corporation (Columbia, Md.) produces an ARA-containing fungal oil (ARASCO®; U.S. Pat. No. 5,658,767) which is substantially free of EPA and which is derived from either Mortierella alpina or Pythium insidiuosum. One of the primary markets for this oil is infant formula; e.g., formulas containing Martek's ARA oils are now available in more than 60 countries worldwide.
Despite the availability of ARA from natural microbial sources such as those described above, microbial production of ARA using recombinant means is expected to have several advantages over production from natural microbial sources. For example, recombinant microbes having preferred characteristics for oil production can be used, since the naturally occurring microbial fatty acid profile of the host can be altered by the introduction of new biosynthetic pathways in the host and/or by the suppression of undesired pathways, thereby resulting in increased levels of production of desired PUFAs (or conjugated forms thereof) and decreased production of undesired PUFAs. Secondly, recombinant microbes can provide PUFAs in particular forms which may have specific uses. And, finally, microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrate sources for microbially expressed enzymes, or by addition of compounds/genetic engineering to suppress undesired biochemical pathways. Thus, for example, it is possible to modify the ratio of ω-3 to ω-6 fatty acids so produced, or engineer production of a specific PUFA (e.g., ARA) without significant accumulation of other PUFA downstream or upstream products. The latter possibility is of particular interest in some embodiments of the invention herein, wherein it is desirable to provide a recombinant source of microbial oil containing high concentrations of ARA and that is additionally devoid of gamma-linolenic acid (GLA; γ-linolenic acid; cis-6, 9, 12-octadecatrienoic acid; ω-6).
GLA is an important intermediate in the biosynthesis of biologically active prostaglandin from LA. Although also recognized as an essential ω-6 PUFA having tremendous clinical, physiological and pharmaceutical value, there are some applications in which GLA acts in opposition to ARA. Thus, commercial production of an oil comprising ARA and devoid of GLA would have utility in some applications.
Most microbially produced ARA is synthesized via the Δ6 desaturase/Δ6 elongase pathway (which is predominantly found in, algae, mosses, fungi, nematodes and humans) and wherein: 1.) oleic acid is converted to LA by the action of a Δ12 desaturase; 2.) LA is converted to GLA by the action of a Δ6 desaturase; 3.) GLA is converted to DGLA by the action of a C18/20 elongase; and 3.) DGLA is converted to ARA by the action of a Δ5 desaturase (FIG. 1). However, an alternate Δ9 elongase/Δ8 desaturase pathway for the biosynthesis of ARA operates in some organisms, such as euglenoid species, where it is the dominant pathway for formation of C20 PUFAs (Wallis, J. G., and Browse, J. Arch. Biochem. Biophys. 365:307-316 (1999); WO 00/34439; and Qi, B. et al. FEBS Letters. 510:159-165 (2002)). In this pathway, LA is converted to EDA by a Δ9 elongase, EDA is converted to DGLA by a Δ8 desaturase, and DGLA is converted to ARA by a Δ5 desaturase.
Although genes encoding the Δ6 desaturase/Δ6 elongase and the Δ9 elongase/Δ8 desaturase pathways have now been identified and characterized from a variety of organisms, and some have been heterologously expressed in combination with other PUFA desaturases and elongases, neither of these pathways have been introduced into a microbe, such as a yeast, and manipulated via complex metabolic engineering to enable economical production of commercial quantities of ARA (i.e., greater than 10% with respect to total fatty acids). Additionally, considerable discrepancy exists concerning the most appropriate choice of host organism for such engineering.
Recently, Picataggio et al. (WO 2004/101757 and co-pending U.S. Patent Application No. 60/624,812) have explored the utility of oleaginous yeast, and specifically, Yarrowia lipolytica (formerly classified as Candida lipolytica), as a preferred class of microorganisms for production of PUFAs such as ARA and EPA. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation can be up to about 80% of the cellular dry weight. Despite a natural deficiency in the production of ω-6 and ω-3 fatty acids in these organisms (since naturally produced PUFAs are limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids)), Picataggio et al. (supra) have demonstrated production of 1.3% ARA and 1.9% EPA (of total fatty acids) in Y. lipolytica using relatively simple genetic engineering approaches and up to 28% EPA using more complex metabolic engineering. However, similar work has not been performed to enable economic, commercial production of ARA in this particular host organism.
Applicants have solved the stated problem by engineering various strains of Yarrowia lipolytica that are capable of producing greater than 10-14% ARA in the total oil fraction, using either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway (thereby producing 10-11% ARA-oil with 25-29% GLA or 14% ARA-oil that is devoid of GLA, respectively). Additional metabolic engineering and fermentation methods are provided to further enhance ARA productivity in this oleaginous yeast.